Project Summary Medicines that cause G protein?coupled receptors (GPCRs) to selectively stimulate arrestins, or to selectively avoid stimulation of arrestins, promise more effective and safer treatments for a wide variety of diseases, including neuropsychiatric, cardiovascular, pulmonary and metabolic disorders. Despite intense study of GPCR?arrestin interactions in both academia and the pharmaceutical industry, and despite dramatic recent advances in the structural biology of GPCRs and arrestins, the mechanism by which GPCRs stimulate arrestins remains poorly understood. Likewise, the means by which GPCRs might achieve selectivity for or against arrestin signaling remains unclear. The proposed research will utilize atomic-level molecular dynamics simulations to address these challenges, thereby providing a foundation for the design of functionally selective GPCR-targeted drugs with desired effects on arrestins. Aim 1 is to determine the activation mechanism of arrestin, pinpointing which of the GPCR? arrestin interaction surfaces drives arrestin activation and discovering the allosteric coupling between regions of arrestin that causes these structural changes to take place. The remaining aims are to determine the effect of both GPCR conformation (Aim 2) and GPCR phosphorylation pattern (Aim 3) on arrestin binding and activation. This will reveal how a GPCR can favor or disfavor arrestin recruitment and signaling relative to G protein recruitment and signaling. It will also reveal how a GPCR can favor specific arrestin conformations, potentially stimulating some of arrestin?s downstream effects without stimulating others. The proposed research will rely on state-of-the-art simulation methods that have recently enabled the determination of functional mechanisms of GPCRs, G proteins, transporters, and other proteins. It will also benefit from close collaborations with multiple experimentalists: results from crystallography, fluorescence spectroscopy, NMR, electron paramagnetic resonance, and cell signaling experiments will combine to both guide and validate the simulations. This proposal is significant not only because it will illuminate a quintessential biological signaling process but also because it will reveal a key part of the structural basis for functional selectivity at GPCRs. It will thus provide a foundation for the rational design of safer and more effective medications acting at GPCRs, which are by far the largest class of drug targets.